Physical/mechanical and antibacterial properties of orthodontic adhesives containing Sr-bioactive glass nanoparticles, calcium phosphate, and andrographolide

White spot lesions around orthodontic brackets are the major complication during fixed orthodontic treatment. This study prepared orthodontic adhesives for promoting mineral precipitation and reducing bacterial growth. Adhesives with added calcium phosphate monohydrate/Sr-bioactive glass nanoparticles (Sr/CaP) and andrographolide were prepared. The physical/mechanical and antibacterial properties of the adhesives were tested. The additives reduced the monomer conversion of the materials (62 to 47%). The addition of Sr/CaP and andrographolide increased the water sorption (from 23 to 46 μg/mm3) and water solubility (from 0.2 to 5.9 μg/mm3) but reduced the biaxial flexural strength (from 193 to 119 MPa) of the adhesives. The enamel bond strengths of the experimental adhesives (19–34 MPa) were comparable to that of the commercial material (p > 0.05). The Sr/CaP fillers promoted Ca, Sr, and P ion release and the precipitation of calcium phosphate at the debonded interface. An increase in the Sr/CaP concentration enhanced the inhibition of S. mutans by 18%, while the effect of andrographolide was not detected. The abilities of the adhesives to promote ion release, calcium phosphate precipitation, and the growth inhibition of cariogenic bacteria were expected to reduce the occurrence of white spot lesions. The additives reduced the physical/mechanical properties of the materials, but the corresponding values were within the acceptable range.

The common complication during fixed orthodontic treatments is initial enamel caries or white spot lesions around the brackets 1 . These lesions occur due to the continuation of tooth demineralization and the dysbiotic dental biofilm 2 caused by suboptimal oral hygiene during the treatment. If the carious lesions are left untreated, the lesions may progress and become deep and uncleanable cavities. This may subsequently lead to severe tooth infection/pain, complicating the orthodontic treatment. It was reported that the irregular surfaces of excess orthodontic adhesives promoted the accumulation of dental biofilms 3 . Hence, adhesives that offer remineralization and antibacterial actions may be needed to reduce the risk of dental caries. However, the currently available and commonly used resin composite orthodontic adhesives showed no anti-caries actions. Additionally, a suboptimal light-curing technique may increase the risk of releasing unreacted monomers 4 , which may promote the cariogenicity and microbial dysbiosis of dental biofilms 5 .
Various methods have been employed to enhance the remineralization and antibacterial actions of orthodontic adhesives. For example, glass ionomer cements were employed to facilitate fluoride release, enhancing caries resistance via the fluoridation of the tooth surface 6 . However, a clinical study reported that a significant Preparation of orthodontic adhesives. The experimental orthodontic adhesives were prepared using the protocol of a previous study 12 . Briefly, the liquid phase of the adhesives contained 70 wt% urethane dimethacrylate (Sigma-Aldrich, St. Louis, MO, USA), 26 wt% triethyleneglycol dimethacrylate (Sigma-Aldrich), 3 wt% 2-hydroxyethyl methacrylate (Sigma-Aldrich), and 1 wt% camphorquinone (Sigma-Aldrich). The powder phase contained silane-treated boroaluminosilicate glass (Esstech, Inc. Essington, PA, USA), Sr-bioactive glass nanoparticles (Sr-BGNPs), monocalcium phosphate monohydrate (MCPM, Himed, Old Bethpage, NY, USA), and andrographolide (Nanjing NutriHerb BioTech, Jiangsu, China). Five experimental formulations with varying concentrations of Sr-BGNPs/MCPM (Sr/Ca) and Andro were prepared ( Table 1). The powder and liquid phases were mixed using a powder to liquid ratio of 4:1 (mass ratio). The mixed adhesives were then loaded in a composite syringe (MIXPAC 1 mL syringe, medmix Switzerland AG, Haag, Switzerland). Commercial resin composite orthodontic adhesive (Trans, Transbond XT, 3M-ESPE, Seefeld, Germany) was used as a control.
Degree of monomer conversion. The uncured adhesives (n = 5) were placed in a metal circlip (1 mm in thickness and 10 mm in diameter) over the diamond of attenuated total reflection (ATR, iD7 ATR, Thermo Fisher Scientific, Waltham, MA, USA) of a Fourier transform infrared spectroscope (FTIR, Nicolet iS5, Thermo www.nature.com/scientificreports/ Fisher Scientific). The materials were then covered with an acetate sheet and light-activated for 20 s using an LED light-curing unit (irradiance of 1200 mW/cm 2 , SmartLite Focus Pen Style, DENTSPLY Sirona, York, PA, USA) at a distance of 1-2 mm from the top of the surface of each material. The FTIR spectra in the region of 700-4000 cm −1 were recorded from the bottom of each material. The degree of monomer conversion (DC) was then calculated using the following equation 12 .
where A 0 and A t are the absorbance of the C-O peak (1320 cm −1 ) 28 above the baseline at 1335 cm −1 before and after curing at time t, respectively.
Biaxial flexural strength and modulus of elasticity. Disc specimens were prepared (n = 8). The composites were placed in a metal ring (10-mm in diameter and 1-mm in thickness). The materials were covered with an acetate sheet and glass slides on the top and bottom sides. The specimens were cured by an LED lightcuring unit for 20 s on both sides. They were left for 24 h at 25 ± 1 °C. Then, the specimens were placed into 10 mL of deionized water at 37 °C. The biaxial flexural strength (BFS) test was performed using a ball-on-ring testing jig with a universal testing machine (AGSX, Shimadzu, Kyoto, Japan). The specimen was loaded with a 500 N load cell with a crosshead speed of 1 mm/min until the specimen failed. The BFS (Pa) was calculated using the following equation 29 .
where F is the failure load (N), d is the thickness of the sample (m), r is the radius of circular support (mm), and a is Poisson's ratio (0.3). Additionally, the biaxial flexural modulus (BFM, Pa) was calculated using the following equation.
where H W c is the rate of change of the load with regard to the central deflection or gradient of force versus the displacement curve (N/m), β c is the center deflection junction (0.5024), and q is the ratio of the support radius to the radius of the disc.
Water sorption and solubility. Disc specimens (n = 6) were prepared and weighed using a four-figure balance. The test was performed according to BS EN ISO 4049 30 . The disc specimens were placed in a desiccator and kept in an incubator at the controlled temperature of 37 ± 1 °C for 22 h. Then, the desiccator was kept at room temperature (25 ± 1 °C) for ~ 2 h. The weight of each specimen was measured until a constant mass (m 1 ) was obtained. The specimens were then placed in 10 mL of deionized water at 37 ± 1 °C for up to 4 weeks. Each specimen's mass was then recorded (m 2 ). Specimens were then reconditioned following the steps described above for m 1 until a constant mass was obtained (m 3 ). The water sorption (W SP , g/m 3 ) and water solubility (W SL , g/m 3 ) were calculated using the following equations.
where m 1 is the conditioned mass of the specimen (g), m 2 is the mass of the specimen after immersion in water for 4 weeks (g), m 3 is the reconditioned mass of the specimen after immersion in water (g), and v is the volume of the specimen (m 3 ).  Table 1. Formulations of the experimental orthodontic adhesives.

Enamel shear bond strength (SBS) and adhesive remnant index (ARI score
Component/formulations S10A10 (wt%) S10A5 (wt%) S5A10 (wt%) S5A5 (wt%) S0A0 (wt%) The extracted teeth were kept in 0.1% thymol solution at room temperature for less than 30 days prior to the test (n = 5). The root was cut at 2 mm under the cervical line. The buccal surface was conditioned with 37% phosphoric acid (Transbond™ XT etching gel; 3 M-ESPE) and rinsed for 15 s followed by gentle air-drying. The etched surface received a primer application (Transbond™ XT Light Cure Orthodontic Primer, 3 M-ESPE) for 10 s and was then air-dried. The experimental adhesives were then applied on the primed surface, followed by the placement premolar brackets (GEMINI MBT 0.022 Twin, 3 M-ESPE). The excess adhesive was removed. Then, the specimen was light-cured using an LED light-curing unit for 10 s on the mesial and distal sides. Specimens were embedded in a self-cured acrylic resin in a polyvinyl chloride (PVC) tube and immersed in artificial saliva 31 for 24 h. Then, the specimens were subjected to thermocycling (5 and 55 °C) with an immersion time and dwell time of 30 s and 10 s for 500 cycles according to PD ISO/TS 11405:2015 32 . Then, the specimens were placed in a shear-bond-strength testing jig under a mechanical testing frame. A knife-edge chisel was applied at the interface between the tooth and the bracket. The specimens were loaded with a 500 N load cell at a crosshead speed of 1 mm/min. The maximal load (F, Newton) before the debonding of the bracket was recorded. The shear bond strength of materials to enamel (SBS, Pa) was then calculated using the following equation 12 .
where A is the area of the bonding interface (m 2 ). An adhesive remnant index was determined by observing the residual adhesive on the brackets under a stereomicroscope (10 × magnification). The ARI index was classified into four categories as follows 33,34 .
• Score 0: no adhesive remained on the enamel. • Score 1: less than 50% of the adhesive remained on the enamel surface.
• Score 2: more than 50% of the adhesive remained on the enamel surface.
• Score 3: all adhesive remained on the enamel surface.
Calcium phosphate precipitation. The test was performed according to the protocol used in the previous study 12 . The specimen (n = 1) was prepared according to the SBS test. The specimens were placed in 10 mL of artificial saliva at 37 °C for 24 h. Then, the brackets were de-bonded from the tooth. The surface of detached bracket was then coated with Au using a sputter-coating machine with a current of 23 mA for 45 s. The precipitation of calcium phosphate crystals on the surface was assessed under a scanning electron microscope (SEM, JSM, 7800F, JEOL Ltd., Tokyo, Japan). Then, the elemental composition of the precipitation was analyzed using an energy dispersive X-ray analysis (EDX, X-sight 6650 detector, Oxford Instruments, Abingdon, UK) with a beam voltage set at 10 kV.
Ion release. Disc specimens (n = 3) were prepared and placed in 10 mL of deionized water. The tubes were incubated at 37 °C for 4 weeks. The storage solution was then collected to analyze the concentrations of Ca, P, and Sr ions. The extract was mixed with 3 vol% nitric acid. The concentrations of ions were assessed using inductively coupled plasma-atomic emission spectroscopy (ICP-OES, Optima 8300, PerkinElmer, Waltham, MA, USA).

Influence on the growth of S. mutans. Streptococcus mutans (ATCC 25175) was inoculated in Mueller
Hinton (MH) broth (BD Difco™ Mueller Hinton Broth, Thermo Fisher Scientific Inc., Göteborg, Sweden) using a 1:2 volume ratio of inoculum to broth 12 . They were incubated for 24 h at a controlled temperature of 37 °C and enriched with 5% CO 2 . The suspension of S. mutans was then adjusted until a bacterial concentration of 2.5 × 10 5 cells/mL was obtained using a spectrophotometer at an optical density (OD) of 600 nm.
Disc specimens (1 mm in thickness and 10 mm in diameter) were prepared and sterilized through UV irradiation for 30 min on each surface (n = 3) 35 . The discs were then placed in tubes containing mixtures of 2 mL of Mueller Hinton Broth and 1 mL of the suspension of S. mutans. A tube without a disc specimen was used as the blank control. The tubes were incubated at a controlled temperature of 37 °C in air enriched with 5% CO 2 for 48 h. Then, the discs were removed. The suspensions were vortexed for 30 s, followed by serial dilution until bacterial concentrations of 1 × 10 -6 CFU/mL were obtained. The suspensions (200 µL) were then plated on Mitis Salivarius agar and incubated at 37 °C under a 5% CO 2 atmosphere for 48 h. Colony-forming units (log CFU/ mL) were then counted using a microscope and image analysis.
Statistical analysis. Numerical data are reported as the mean and SD. The results were analyzed using Prism version 9.3 for macOS (GraphPad Software, San Diego, CA, USA). The normality of the data distribution was evaluated using the Shapiro-Wilk test. BFS and ion release results were compared using one-way ANOVA followed by Tukey's multiple comparisons test. DC, BFM, W sp , W SL , SBS, and antibacterial tests were compared using the Kruskal-Wallis test followed by the Dunn test. A chi-squared test was used to evaluate the ARI scores among the adhesive subgroups. Statistical significance was set at p = 0.05. The sample size used in each test was calculated by G*Power 3.1 software (University of Dusseldorf, Dusseldorf, Germany) using the results in published studies and a pilot study 10,12,29 . The result indicated that the sample size in each test gave power > 0.95 at www.nature.com/scientificreports/ alpha = 0.05. Additionally, factorial analysis was performed to assess the effects of increasing the concentrations of Sr/CaP (5 to 10 wt%) and Andro (5 to 10 wt%) on the tested properties 10 .

Characterization of Sr-bioactive glass nanoparticles (SrBGNPs). Monodisperse spherical Sr-
BGNPs were successfully synthesized in the 170 ± 30-nm-diameter range through a sol-gel process (Fig. 1A,B). The elemental composition of the Sr-BGNPs was 81.0 mol% SiO 2 , 14.2 mol% CaO, and 4.8 mol% SrO. The XRD pattern of the Sr-BGNPs showed a broad halo of particles calcined at 680 °C, indicating that calcium oxide (CaO) and strontium oxide (SrO) were successfully incorporated into the amorphous structure (Fig. 1C).

Enamel shear bond strength (SBS) and adhesive remnant index (ARI score). The highest and
lowest SBSs (median, min-max) were obtained from S0A0 (34.6, 24.9-41.5 MPa) and S10A10 (18.0, 11.0-27.1 MPa) (Fig. 3A). The SBS of Trans (26.6, 23.5-40.8 MPa) was similar to those of S10A10, S10A5 (17. The factorial analysis demonstrated that an increase in Sr/CaP from 5 to 10 wt% reduced SBS by 28 ± 22%. The effect of Andro was negligible. The most common ARI scores observed from all materials were scores of 0 and 1 (Fig. 3B). The ARI scores of 2 and 3 were not observed from the specimens. The distribution of the score among each group was not similar (p < 0.05).
Calcium phosphate precipitation. The precipitation of calcium phosphate crystals was detected on the debonded surfaces of the brackets of all groups except for S0A0 and Trans. The EDX results demonstrated that the precipitate contained Ca and P (Fig. 4).
Ion release. Ca, P, and Sr ions were not detected from S0A0 and Trans. The highest and lowest concentrations of Ca ions were detected from S10A5 (1.70 ± 0.29 ppm) and S5A5 (0.61 ± 0.02 ppm) (Fig. 5A). For P, the www.nature.com/scientificreports/ highest and lowest concentrations were detected from S10A5 (3.44 ± 0.10 ppm) and S5A5 (1.22 ± 0.03 ppm) (Fig. 5B). Likewise, the highest and lowest Sr concentrations were detected from S10A5 (1.99 ± 0.73 ppm) and S5A5 (0.85 ± 0.22 ppm) (Fig. 5C). The factorial analysis demonstrated that an increase in Sr/CaP from 5 to 10 wt% increased the amounts of released Ca and P by 141 ± 19% and 119 ± 4%, respectively. The effects of increasing the concentrations of Sr/CaP and Andro on the release of Sr were negligible.

Discussion
The limitation of the commonly used resin-based orthodontic adhesive is the lack of ion release and antibacterial actions resulting in the formation of white spot (early caries) lesions around brackets, which is a common esthetic complication during orthodontic treatment. The aim of the current study was to prepare experimental orthodontic adhesives containing Sr/CaP and Andro for promoting ion release and antibacterial action, which could potentially help reduce the risk of white spot lesions. The effects of Sr/CaP and Andro on the physical/ mechanical and antibacterial actions of the adhesives were then determined. The research hypothesis was rejected because increases in the concentrations of additives significantly affected the water sorption/solubility, biaxial www.nature.com/scientificreports/ flexural strength and modulus, shear bond strength, mineral precipitation, ion release, and growth of S. mutans of the experimental adhesives. High degrees of monomer conversion of orthodontic adhesives are required to reduce the risk of toxic monomer leaching and provide sufficient adhesive mechanical strength 36 . The release of unbound monomers due to the suboptimal polymerization of orthodontic adhesives could potentially enhance the cariogenicity of dental biofilms 5 . The additives reduced the DC of the experimental orthodontic adhesives, which could be due to an increase in refractive index mismatch 10 . This may increase light scattering and reduce light penetration and polymerization at the inner surface. The higher DCs of the experimental materials compared to that of Trans could be due to the differences in the primary methacrylate monomer. Bisphenol A-glycidyl methacrylate (Bis-GMA) is the primary base monomer of Trans. The glass transition temperature (T g ) of UDMA (− 35.3 °C), which was the primary base monomer in the experimental material, is lower than that of Bis-GMA (− 7.7 °C). The polymer containing monomers with a low glass transition temperature (T g ) usually attains a higher level of monomer conversion compared to polymers consisting of high-T g monomers 37,38 . A higher monomer conversion was expected to reduce the risk of monomer release 39 . However, elution studies and toxicity tests should be included in future work.
Resin-based materials can absorb water from the environment, leading to hygroscopic expansion, hydrolytic degradation, and the release of the active components. Excessive water sorption may cause water plasticization, which could subsequently reduce the strength of the materials 40,41 . Additionally, water sorption may lead to the volume expansion of resin-based material, which could help compensate for the polymerization shrinkage stress that occurred within the material 42 . The W sp values of S10A10 and S10A5 were higher than the level required by BS ISO 4049 (< 40 µg/mm 3 ). This could be due to the hydrophilicities of MCPM and the Sr-BGNPs. However, the W sp values were much lower than those of previously developed Ca/P-releasing orthodontic adhesives containing 10 wt% MCPM 12 . This could be because the concentration of MCPM (2.5-5 wt%) used in the current study was lower than that used in the previous study. The water sorption values of all experimental adhesives were within the range of that required by BS ISO 4049 for luting-type dental composites (< 7.5 µg/mm 3 ). Previous studies reported that the addition of MCPM promoted ion release and the formation of calcium phosphate fillers, such as dicalcium phosphate dihydrate (DCPD), the structure of which contains water 43,44 . The formation of dicalcium phosphate in the materials may help decrease mass loss and water dissolution. Other possible explanations include the slow degradation of strontium-bioactive glass nanoparticles and the low water solubility of andrographolide.
Currently, there are no minimum requirements specified by the ISO for the enamel shear bond strengths of orthodontic adhesives. A high SBS is essential for ensuring that the active force from the archwire can be transferred to the anchored tooth without dislodgement. Although the SBSs of the experimental adhesives were lower than that of Trans, the values were still higher than the minimum clinically acceptable SBS (5.9-7.8 MPa) 45 . It is speculated that the addition of nonsilanized fillers may detrimentally reduce the SBSs of the experimental adhesives by the accelerated degradation of the matrix-filler interface during thermocycling. The SBSs of the experimental adhesives obtained from the experimental materials were still within the range or higher than that reported in published studies   46,47 . This could be due to the use of a minimum number of aging cycles (500 cycles) according to the ISO (British Standard PD ISO/TS 11405:2015 Dentistry-Testing of adhesion to tooth structure) in the current study 32 . A larger number of aging cycles, i.e., 5,000-30,000 cycles, should be used in future studies to ensure the long-term bonding performance of the materials 48 .
Although the observed SBS values among the experimental adhesives were similar, the factorial analysis indicated that the incorporation of Sr/CaP reduced the SBSs of the materials by ~ 28%. The reduction in SBS upon the addition of reactive fillers could be due to the lack of silanization of the additives or the hydrophilicity of the Sr/CaP fillers 49,50 . It is speculated that the main effect could be primarily from MCPM due to its high water solubility (~ 18 g/L at 25 °C) 11,44,50,51 . Hence, future work may need to assess the formulation with low-level MCPM (1-2 wt%) or no MCPM (0 wt%). This could additionally help reduce the excessive water sorption/ solubility of the experimental materials.
The adhesive remnant index (ARI, 0 to 4) is one of the most commonly used methods for assessing the characteristics of the adhesion between the adhesive and enamel. A high ARI score may indicate excessively strong adhesion between the adhesive and enamel surface, which could lead to enamel fracture at the interface. Hence, failure within the adhesive layer (low ARI score) during the detachment of brackets is preferred to reduce the risk of the breakdown of the enamel surface. The ARI scores of the materials in the current studies were mostly 0 or 1, which was similar to that of the published studies 52,53 . This may be desirable to help preserve the enamel surface during the debonding of brackets.
The addition of Sr/CaP fillers promoted the release of Ca, P, and Sr ions. These ions were expected to promote suitable conditions for the precipitation of biological hydroxyapatite to enhance remineralization. The experimental adhesives containing high Sr/CaP concentrations (S10A10, S10A5) showed greater amounts of released Ca and P ions compared to formulations with lower Sr/CaP concentrations. A possible explanation for this could be that the release of Ca and P ions was primarily due to the dissolution of MCPM. The differences in Sr 2+ -release among groups were not clearly observed, which may be due to the lower solubility of the Sr-BGNPs. All experimental adhesives containing Sr/CaP promoted the precipitation of calcium phosphate at the interface. The EDX results showed that the Ca/P atomic ratio of the precipitate (0.5) on the debonded brackets was much lower than that of hydroxyapatite (1.67). This may suggest that the precipitate could be representative of the early stage of calcium phosphate crystal formation because the immersion time of the specimen was only 24 h. The Ca/P ratio may be increased with an increase in the immersion time 54 . A previous study demonstrated that the precipitation of hydroxyapatite could be detected on an orthodontic adhesive containing bioactive glass after immersion in artificial saliva for 6 months 55 . A longer immersion time to confirm the ability of the materials to promote apatite formation may be employed in future studies. www.nature.com/scientificreports/ The increase in the level of Sr/CaP from 5 to 10 wt% (S10A10, S10A5) enhanced the growth inhibition of the planktonic S. mutans exhibited by the experimental orthodontic adhesives. It was speculated that the bacteriostatic action observed with the experimental orthodontic adhesives could be primarily due to the Sr-BGNPs since the potential benefits of Sr 2+ -release include both remineralizing and antibacterial actions 18 . This result was in accordance with those of previous studies that demonstrated the antibacterial actions of Sr-containing bioactive glasses 56,57 . It was speculated that the growth inhibition of S. mutans could be due to the release of Sr-BGNPs from the experimental adhesives due to the lack of silanization. The large surface-to-volume ratio and high charge density of the nanoparticles may facilitate interactions with negatively charged bacterial cell membranes, thereby increasing the antimicrobial activity of the nanoparticles even at low concentrations 58 . The current study, however, failed to demonstrate the growth inhibition of planktonic S. mutans by Andro. A possible explanation for this could be due to the low solubility of Andro 59 , which may limit its release from the adhesives. Additionally, it was proposed that the antibacterial action of Andro mainly involved the inhibition of the biofilm through Andro acting as a quorum-sensing inhibitor that interfered with the biofilm-forming process 60 . Furthermore, it should be mentioned that the bacterial growth inhibition may be due to the unreacted monomers released from the materials 61 . Hence, the monomer elution study should be examined in future work.
It should be mentioned that the current study was an in vitro study, so the clinical relevance should be carefully interpreted. The results from this preliminary study indicate that the formulation with a high level of Sr/ CaP (S10A10 and S10A5) generally exhibited desirable and acceptable results except for water sorption/solubility. Further characterization and modification of the formulations may be needed in future works.

Conclusion
Experimental orthodontic adhesives containing MCPM/Sr-BGNPs and Andro were developed for promoting ion release and inhibiting cariogenic bacteria. The additives reduced the physical and mechanical properties of the materials, but the corresponding values were still within the acceptable range. The addition of Sr/CaP promoted calcium phosphate precipitation and the inhibition of planktonic S. mutans. The increase in Andro reduced the strengths of the materials and failed to demonstrate antibacterial actions. These promising properties of the experimental orthodontic adhesives were expected to help reduce the risk of developing white spot lesions around excessive amounts of adhesive for high caries-risk patients.

Data availability
The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request. www.nature.com/scientificreports/